The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
In biospecific binding assays, such as, e.g. immunoassays, nucleic acid hybridization assays, receptor-binding assays, and cellular binding assays, the analytes to be measured are generally present at very low concentrations. Various labelling compounds have been developed that allow labelling reactants to be detected and quantitated at high sensitivity. Time-resolved luminescence spectroscopy using lanthanide chelates has for several years been applied in immunoassays and nucleic acid hybridization assays [e.g. I. Hemmilä, T. Stålberg, and P. Mottram (eds.), “Bioanalytical Applications of Labelling Technologies”, Wallac, Turku, 1994 and D. Wild (eds), “The Immunoassay Handbook”, Nature Publishing Group, 2001]. Stable photoluminescent (referred in the context of this specification simply as luminescent) lanthanide chelates have also other applications, e.g. fluorescence microscopy and cytometry. Therefore, a number of attempts have been made to develop new highly luminescent chelates suitable for those types of time-resolved fluorometric applications. These include e.g. stable chelates composed of derivatives of pyridine (U.S. Pat. No. 4,920,195; U.S. Pat. No. 4,801,77; U.S. Pat. No. 4,761,481; Bush. C. E. et al. 1992, Anal. Biochem., 202,146; WO 92/14841; Hemmilä, et al., 1993, J. Biochem. Biophys. Methods, 26, 283; U.S. Pat. No. 5,571,897; U.S. Pat. No. 5,859,215, Latva, M. et al., 1997, J. Luminescence, 75, 149; Takalo, H. et al., 1996, Helv. Chim. Acta, 79, 789; von Lode, P. et al., 2003, Anal. Chem. 75, 3193; U.S. Pat. No. 7,018,851; WO 2005/021538; US 2005/0181393); bipyridines (U.S. Pat. No. 5,216,134), terpyridines (U.S. Pat. No. 4,859,777; U.S. Pat. No. 5,202,423; U.S. Pat. No. 5,324,825) or various phenolic compounds (U.S. Pat. No. 4,670,572; U.S. Pat. No. 4,794,191; IT 1235668) as the energy mediating groups and polycarboxylic acids as chelating parts. In addition, various dicarboxylate derivatives (U.S. Pat. No. 5,032,677; U.S. Pat. No. 5,055,578; U.S. Pat. No. 4,772,563), macrocyclic cryptates (U.S. Pat. No. 4,927,923; WO 93/5049; EP 0 493 745), calixarenes (Sato, N. et al., 1993, J. Chem. Soc. Perkin Trans. 2, 621; Steemers, F. J. et al., 1995, J. Am. Chem. Soc., 117, 9408), DTPA carbostril 124 conjugate (Selvin, P. R., et al., 1994, J. Am. Chem. Soc., 116, 6029) and macrocyclic Schiff bases (EP 0 369 000) have been disclosed in patent applications and/or patents.
Since the publication of chelates, which contain two and three separate 4-(phenylethynyl)pyridine moieties (Takalo, H. et al., Helv. Chim. Acta, 79, 789) the designed chelating structures have been applied in patent applications and publications where the phenylethynyl substituents have been replaced with 2-furyls and trialkoxyphenyls (WO 2005/021538; US 2005/0181393, Jaakkola, L. et al. 2005, Bioconjugate Chem., 16, 700). Moreover, by combining two reactive functional groups for coupling the label to a biospecific binding reactant highly luminescent chelates have been obtained (U.S. Pat. No. 7,018,851). Chelates with 2,4,6-trimethoxypyridine chromophores have shown high luminescence especially with Tb(III) and Dy(III) ions (U.S. Pat. No. 4,761,481; Hemmilä, et al, 1993, J. Biochem. Biophys. Methods, 26, 283; Latva, M. et al., 1997, J. Luminescence, 75, 149, US 2005/0181393; Jaakkola, L. et al. 2005, Bioconjugate Chem., 16, 700). The triplet-state energy level of the ligand, the nature of the chromophore and the biomolecule binding group have significant effect on the luminescent properties of the Tb(III) labels (see e.g. Sabbatini, N. et al., 1991, J. Luminescence 48 & 49: 463; Hemmilä, et al., 1997, J. of Alloys and Compounds 249, 158). Thus even small changes in chelate structure may significantly reduce luminescence of Tb(III) labels.
It is known that the addition of chromophores decreases the solubility of ligands and chelates in water, increases the formation of biospecific binding reactant aggregates during the labelling process and non-specific binding properties of labelled biomolecules. Aggregates will produce purification problems and reduced yield of labelled material. Moreover, increased non-specific binding of labelled biomolecule will enhance background luminescence of biospecific assays and thus reduces assay sensitivity.